Type 2 diabetes is the most prevalent form of diabetes in the United States. The major contributing factor to the development of type 2 diabetes is insulin resistance of peripheral tissues, primarily skeletal muscle and adipose tissue. Insulin mediates the translocation and fusion of insulin-responsive glucose transporter (GLUT4)-containing vesicles to the plasma membrane;this process is critical for proper glucose homeostasis. Thus, the identification and characterization of cellular factors and processes regulating GLUT4 function are critical to understanding the molecular mechanisms underlying impaired insulin sensitivity. While insulin signaling coordinates the translocation and fusion of GLUT4 vesicles by regulating actin reorganization and cytoskeletal remodeling little is known about the cellular contractile forces required for these processes. The broad aim of this proposal is to gain insight on the mechanisms regulating the dynamic reorganization of the cytoskeleton during insulin-stimulated GLUT4 vesicle trafficking. The myosin family of protein, specifically myosin II has been shown to regulate actin filament reorganization to facilitate vesicle trafficking. Our preliminary studies show that the myosin IIA isoform is activated and recruited to the plasma membrane upon insulin stimulation to facilitate GLUT4-mediated glucose uptake. We also have found that insulin regulates myosin IIA via myosin light chain kinase (MLCK), a Ca2+ regulated kinase to promote myosin IIA activation and recruitment to the plasma membrane. Furthermore, we show that inhibition of myosin II activity impairs the proper insertion of GLUT4 at the plasma membrane. Previous studies have shown a requirement for intracellular Ca2+ for insulin stimulated GLUT4 translocation and fusion with the plasma membrane. Based on our findings, we hypothesize that Ca2+ mediates the activation and recruitment of myosin IIA to the cell cortex to facilitate GLUT4 fusion with the plasma membrane. Thus, the specific aims of this proposal are to define and characterize the role of Ca2+ on the regulation of myosin IIA in GLUT4 vesicle trafficking in adipocytes, and to determine the mechanisms by which myosin IIA regulates actin reorganization. PUBLIC HEALTH RELEVANCE: Type 2 diabetes is a major public health issue in the United States affecting approximately 6% of the population. The prevalence of type 2 diabetes is even higher among African Americans, Hispanics, Native Americans, Asian Americans, and the elderly. A particularly alarming trend is the recent dramatic rise in the incidence of type 2 diabetes in children. With the increasing incidence of type 2 diabetes it is critical to identify factors contributing to the development of this disease in order to design effective treatments. The proposed research will provide critical information for understanding the molecular mechanism(s) involved in the development of in type 2 diabetes.